Indirubin-3'-oxime inhibits c-Jun NH2-terminal kinase: anti-apoptotic effect in cerebellar granule neurons

Previous studies have demonstrated that c-Jun NH2-terminal protein kinase (JNK) plays a crucial role in neuronal apoptosis. Here, we report that indirubin-3'-oxime, a known effective inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3-beta (GSK-3beta), has a significant inhibitory effect on JNK. Kinase assay showed that indirubin-3'-oxime directly inhibited the activity of all three isoforms of JNK (JNK1, and JNK3) in vitro, with half inhibition dose (IC50) of 0.8 microM, 1.4 microM, and 1.0 microM, respectively. In cerebellar granule neurons (CGNs), indirubin-3'-oxime blocked c-Jun phosphorylation induced by potassium withdrawal and prevented CGNs from apoptosis in a dose dependent manner. However, inhibitors of CDKs and GSK-3beta were ineffective in reducing c-Jun phosphorylation both in vitro and in vivo, suggesting that indirubin-3'-oxime prevents c-Jun phosphorylation independent of its inhibition on CDKs and GSK-3beta. Our studies give further supports for JNK-targeting strategy in preventing neuronal apoptosis.     

Glutathione S-transferase pi regulates UV-induced JNK signaling in SH-SY5Y neuroblastoma cells

Activation of c-Jun N-terminal kinase (JNK) signaling pathway is a key event in apoptosis. The cellular mechanisms underlying the control of JNK catalytic activity before and immediately after stress in neuronal cells are still not completely understood. Under resting conditions the basal activity of JNK is low, since JNK is kept inactive by the presence of one or more endogenous repressors, including glutathione S-transferase pi (GSTpi). The aim of this study was to investigate the control of JNK signaling by GSTpi. We examined the modifications of GSTpi protein expression and oligomerization after UV irradiation-induced stress in human SH-SY5Y neuroblastoma cells. In parallel, we investigated the effect of UV irradiation on JNK activation and c-Jun phosphorylation, and whether apoptosis represents a functional consequence triggered by this signaling pathway. We show that in SH-SY5Y cells JNK phosphorylation and activation precedes c-Jun phosphorylation and caspase-3 cleavage. Importantly, the increase of JNK enzymatic activity correlates with the dissociation of GSTpi–JNK complexes and the increased concentration of GSTpi multimer forms. Results presented herein show for the first time direct interaction between JNK and GSTpi in SH-SY5Y neuroblastoma cells, and suggest that in these cells GSTpi may serve as a regulator of JNK catalytic activity. This work contributes to further elucidate the mechanisms underlying the regulation of JNK activity under stress conditions.     

c-Jun NH2-terminal kinase-mediated signaling is essential for Pseudomonas aeruginosa ExoS-induced apoptosis

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa mainly affects immunocompromised individuals as well as patients with cystic fibrosis. In a previous study, we showed that ExoS of P. aeruginosa, when injected into host cells through a type III secretion apparatus, functions as an effector molecule to trigger apoptosis in various tissue culture cells. Here, we show that injection of the ExoS into HeLa cells activates c-Jun NH(2)-terminal kinase (JNK) phosphorylation while shutting down ERK1/2 and p38 phosphorylation. Inhibiting JNK activation by expression of a dominant negative JNK1 or with a specific JNK inhibitor abolishes ExoS-triggered apoptosis, demonstrating the requirement for JNK-mediated signaling. Following JNK phosphorylation, cytochrome c is released into the cytosol, leading to the activation of caspase 9 and eventually caspase 3. Although c-Jun phosphorylation is also observed as a result of JNK activation, ongoing host protein synthesis is not essential for the apoptotic induction, suggesting that c-Jun- or other AP-1-driven activation of gene expression is dispensable in this process. Therefore, ExoS has opposing effects on different cellular pathways that regulate apoptosis: it shuts down host cell survival signal pathways by inhibiting ERK1/2 and p38 activation, and it activates proapoptotic pathways through activation of JNK1/2 leading ultimately to cytochrome c release and activation of caspases. These results highlight the modulation of host cell signaling by the type III secretion system during interaction between P. aeruginosa and host cells.     

Extracellular signal-regulated kinases are not involved in activity-dependent survival or apoptosis in cerebellar granule neurons

Cerebellar granule neurons (CGNs) depend on potassium depolarization for survival and undergo apoptosis when deprived of depolarizing concentration of potassium. Extracellular signal-regulated kinases (ERK1/2) are thought to be activated in response to potassium depolarization and responsible for the activity-dependent survival in CGNs, but one recent study has revealed that ERK1/2 is activated by potassium deprivation and is required for apoptosis of CGNs. In this study we showed that ERK1/2 was inactivated, rather than activated, by potassium deprivation, indicating a lack of ERK1/2 involvement in potassium deprivation-induced apoptosis. Furthermore, suppression of potassium depolarization-induced activation of ERK1/2 with chemical inhibitor U0126 or PD98059 had no influence on the pro-survival effect of potassium depolarisation. Thus, ERK1/2 was not required for potassium depolarization-dependent survival of CGNs. Taken together, our findings suggest that ERK1/2 is not involved in activity-dependent survival or apoptosis of CGNs.     

Requirement of the JIP1 scaffold protein for stress-induced JNK activation

The c-Jun N-terminal kinase (JNK) signal transduction pathway is activated in response to the exposure of cells to environmental stress. Components of the JNK signaling pathway interact with the JIP1 scaffold protein. JIP1 is located in the neurites of primary hippocampal neurons. However, in response to stress, JIP1 accumulates in the soma together with activated JNK and phosphorylated c-Jun. Disruption of the Jip1 gene in mice by homologous recombination prevented JNK activation caused by exposure to excitotoxic stress and anoxic stress in vivo and in vitro. These data show that the JIP1 scaffold protein is a critical component of a MAP-kinase signal transduction pathway.     

E2F1 is not essential for apoptosis induced by potassium deprivation in cerebellar granule neurons

Cerebellar granule neurons (CGNs) undergo apoptosis when deprived of depolarizing concentration of potassium. A key regulator of cell cycle, E2F1, was believed to play a role in CGN apoptosis induced by potassium deprivation. However, here we demonstrated that although E2F1 was upregulated in wild type CGNs following potassium deprivation, CGNs that derived from E2F1 knockout mice underwent apoptosis at a similar rate as the wild type. Analysis of the apoptotic neurons revealed no difference in the activation of caspase-3 in E2F1 null and wild type CGNs. Furthermore, knockdown of E2F1 expression by RNA interference failed to attenuate the apoptosis of CGNs induced by potassium deprivation. Taken together, our results suggested that E2F1 is not essential for apoptosis induced by potassium deprivation in CGNs.     

Distinct signaling pathways of precursor BDNF and mature BDNF in cultured cerebellar granule neurons

Recent studies have focused on a distinctive contrast between bioactivities of precursor brain-derived neurotrophic factor (proBDNF) and mature BDNF (matBDNF). In this study, using a proteolytic cleavage-resistant proBDNF mutant (CR-proBDNF), signaling mechanisms underlying the proapoptotic effect of proBDNF and antiapoptotic effect of matBDNF on the low potassium (LK)-inducing cell death of cultured cerebellar granule neurons (CGNs) were analyzed. A time course study demonstrated that unlike matBDNF, CR-proBDNF failed to induce TrkB phosphorylation for up to 360 min. CR-proBDNF did not activate ERK-1, ERK-2 and Akt, which are involved in TrkB-induced cell survival signaling, while matBDNF activated these kinases. On the other hand treatment of CGNs with CR-proBDNF led to a rapid activation of Rac-GTPase and phosphorylation of JNK which are involved in p75^NTR-induced apoptosis. In addition, a JNK-specific inhibitor, SP600125, inhibited the CR-proBDNF-induced apoptosis but did not affect the antiapoptotic effect of matBDNF. CR-proBDNF treatment led to an earlier appearance of active caspase-3. In contrast, matBDNF dramatically postponed the appearance of active caspase-3. Not like other signaling molecules, activation of caspase-3 was conversely regulated by both CR-proBDNF and matBDNF. These results thus suggest that in CGNs proBDNF elicits apoptosis via activation of p75^NTR, Rac-GTPase, JNK, and caspase-3, while matBDNF signals cell survival via activation of TrkB, ERKs and Akt, and deactivation of caspase-3.     

The involvement of a stress-activated pathway in equine influenza virus-mediated apoptosis

We have shown elsewhere that equine-2 influenza virus (EIV; subtype H3N8) induced pronounced cell death in infected cells through apoptosis as demonstrated by DNA fragmentation assay and a combined TUNEL and immunostaining scheme. In this study, we investigated the mechanism of EIV-mediated cytotoxicity on a permissive mammalian epithelial cell line, Madin-Darby canine kidney (MDCK) cells. EIV infection increased the cellular levels of oxidative stress and c-Jun/AP-1 protein (which is known to be affected by oxidative stress), as well as its DNA binding activity. Increased production of TGF-beta1, an inducer of c-Jun N-terminal kinase or stress-activated protein kinase (JNK/SAPK) activation, was also detected in EIV-infected MDCK cells. It has been reported that TGF-beta may initiate a signaling cascade leading to JNK/SAPK activation. Addition of c-Jun antisense oligodeoxynucleotide, antioxidant N-acetyl-cysteine (NAC), JNK/SAPK inhibitor carvedilol, or TGF-beta-neutralizing antibody effectively blocked c-Jun/AP-1 upregulation and TGF-beta1 production mediated by EIV infection. These treatments also attenuated EIV-induced cytopathogenic effects (CPE) and apoptosis. Our results suggest that a stress-activated pathway is involved in apoptosis mediated by EIV infection. It is likely that EIV infection turns on the JNK/SAPK cascade, which modulates the activity of apoptosis-promoting regulatory factor c-Jun/AP-1 and epithelial growth inhibitory cytokine TGF-beta.     

Neuroprotection by c-Jun NH2-terminal kinase inhibitor SP600125 against potassium deprivation–induced apoptosis involves the Akt pathway and inhibition of cell cycle reentry

Increasing evidence implicates the c-Jun NH₂-terminal kinase (JNK) pathway in the regulation of apoptosis in neurodegenerative diseases. In this study, we examined the neuroprotective effect of SP600125, a selective JNK inhibitor, in cerebellar granule cells (CGNs) deprived of serum and potassium (S/K withdrawal). S/K withdrawal-induced apoptosis occurs via activation of multiple pro-apoptotic pathways, including re-entry into the cell cycle, activation of glycogen synthase kinase-3 beta (GSK-3β), cyclin-dependent kinase 5 (cdk5/p35) breakdown, formation of cdk5/p25 and JNK activation. Here we demonstrate that SP600125 is able to inhibit all these pro-apoptotic pathways via the inhibition of JNK. Further, we found that JNK inhibition maintains the phosphorylation/activation of Akt after S/K withdrawal. For further confirmation of this result, we studied several targets downstream of Akt including GSK-3β, p-FOXO1, p-CREB and p35. In addition, the specific PI3K/Akt inhibitor LY294002 greatly diminished the antiapoptotic effects of SP600125 upon S/K withdrawal, confirming that Akt is involved in the neuroprotection achieved by SP600125. These results suggest that the maintenance of the PI3-kinase/Akt pathway by inhibition of JNK contributes to the prevention of apoptosis in rat cerebellar granule neurons mediated by S/K withdrawal. Furthermore, we propose that JNK may regulate the cell cycle re-entry by a novel mechanism that involves Akt, GSK-3β and Rb phosphorylation.     

Activation of JNK pathway in persistent pain

The c-Jun N-terminal kinase (JNK) is a stress-activated member of MAP kinase family. JNK activation has been strongly implicated in inflammatory responses, neurodegeneration, and apoptosis. Recent evidence shows that JNK pathway is also transiently activated in primary sensory neurons after tissue or nerve injury, which is required for the development of hyperalgesia and allodynia. In particular, JNK is persistently activated in astrocytes of the spinal cord after nerve injury, and this activation can maintain central sensitization and mechanical allodynia. In this mini-review, we will provide evidence for the involvement of JNK pathway in regulating persistent pain sensitization. We will also discuss possible upstream signaling mechanisms that cause JNK activation and downstream signaling mechanisms by which JNK modulates pain sensitivity. Thus, targeting JNK pathway might be a useful strategy to treat both neurodegeneration and chronic pain.     

JNK and p38 were involved in hypoxia and reoxygenation-induced apoptosis of cultured rat cerebellar granule neurons

As a model of the reperfusion injury found in stroke, we treated cerebellar granule neurons (CGNs) with hypoxia followed by reoxygenation. Hypoxia for 3 h followed by 24 h reoxygenation (H/R) induced a typical apoptosis of CGNs. CGNs exposed to H/R responded by activating JNK, increasing the expression of p38 and ultimately caused CGNs dying. Furthermore, apoptosis of CGNs induced by H/R was inhibited by pre-treatment with SB203580 or SP600125, and the inhibitory effect of SB203580 was greater than that of SP600125. Additionally, we also found that H/R temporally activated Akt and inactivated glycogen synthesis kinase-3β (GSK-3β), two proteins the functions of which were important in cell survival and energy metabolism. These findings demonstrated that H/R-induced apoptosis in CGNs by enhancing JNK and p38 activity, which contributed at least in part to H/R-induced apoptosis of CGNs.     

The CARMA1-Bcl10 signaling complex selectively regulates JNK2 kinase in the T cell receptor-signaling pathway

Members of the c-Jun NH(2)-terminal kinase (JNK) family play crucial roles in cell activation, differentiation, and apoptosis. Although many studies have indicated that JNK1 and JNK2 have functional differences and redundancy, the upstream signaling pathway that selectively activates JNK1 or JNK2 remains unknown. In this study, we have revealed a selective mechanism of JNK activation, in which JNK2, but not JNK1, was regulated by CARMA1, a scaffold molecule, after stimulation of the T cell receptor (TCR). This CARMA1-dependent regulation of JNK2 worked through the scaffold molecule Bcl10, which was inducibly associated with JNK2 and served as a JNK-interacting protein (JIP)-like scaffold to assemble the kinases JNK2, MKK7, and TAK1. Finally, we showed that CARMA1- and Bcl10-mediated JNK2 activation had a critical role in regulating the amount of c-Jun protein. Together, our studies provide genetic evidence that JNK1 and JNK2 are differentially regulated in the TCR-signaling pathway and play different functions.